Assessment of Metabolomic and Proteomic Biomarkers in Detection and Prognosis of Progression of Renal Function in Chronic Kidney Disease

Chronic kidney disease (CKD) is part of a number of systemic and renal diseases and may reach epidemic proportions over the next decade. Efforts have been made to improve diagnosis and management of CKD. We hypothesised that combining metabolomic and proteomic approaches could generate a more systemic and complete view of the disease mechanisms. To test this approach, we examined samples from a cohort of 49 patients representing different stages of CKD. Urine samples were analysed for proteomic changes using capillary electrophoresis-mass spectrometry and urine and plasma samples for metabolomic changes using different mass spectrometry-based techniques. The training set included 20 CKD patients selected according to their estimated glomerular filtration rate (eGFR) at mild (59.9±16.5 mL/min/1.73 m²; n = 10) or advanced (8.9±4.5 mL/min/1.73 m²; n = 10) CKD and the remaining 29 patients left for the test set. We identified a panel of 76 statistically significant metabolites and peptides that correlated with CKD in the training set. We combined these biomarkers in different classifiers and then performed correlation analyses with eGFR at baseline and follow-up after 2.8±0.8 years in the test set. A solely plasma metabolite biomarker-based classifier significantly correlated with the loss of kidney function in the test set at baseline and follow-up (ρ = −0.8031; p<0.0001 and ρ = −0.6009; p = 0.0019, respectively). Similarly, a urinary metabolite biomarker-based classifier did reveal significant association to kidney function (ρ = −0.6557; p = 0.0001 and ρ = −0.6574; p = 0.0005). A classifier utilising 46 identified urinary peptide biomarkers performed statistically equivalent to the urinary and plasma metabolite classifier (ρ = −0.7752; p<0.0001 and ρ = −0.8400; p<0.0001). The combination of both urinary proteomic and urinary and plasma metabolic biomarkers did not improve the correlation with eGFR. In conclusion, we found excellent association of plasma and urinary metabolites and urinary peptides with kidney function, and disease progression, but no added value in combining the different biomarkers data.     

Intergenomic Comparisons Highlight Modularity of the Denitrification Pathway and Underpin the Importance of Community Structure for N2O Emissions

Nitrous oxide (N₂O) is a potent greenhouse gas and the predominant ozone depleting substance. The only enzyme known to reduce N₂O is the nitrous oxide reductase, encoded by the nosZ gene, which is present among bacteria and archaea capable of either complete denitrification or only N₂O reduction to di-nitrogen gas. To determine whether the occurrence of nosZ, being a proxy for the trait N₂O reduction, differed among taxonomic groups, preferred habitats or organisms having either NirK or NirS nitrite reductases encoded by the nirK and nirS genes, respectively, 652 microbial genomes across 18 phyla were compared. Furthermore, the association of different co-occurrence patterns with enzymes reducing nitric oxide to N₂O encoded by nor genes was examined. We observed that co-occurrence patterns of denitrification genes were not randomly distributed across taxa, as specific patterns were found to be more dominant or absent than expected within different taxonomic groups. The nosZ gene had a significantly higher frequency of co-occurrence with nirS than with nirK and the presence or absence of a nor gene largely explained this pattern, as nirS almost always co-occurred with nor. This suggests that nirS type denitrifiers are more likely to be capable of complete denitrification and thus contribute less to N₂O emissions than nirK type denitrifiers under favorable environmental conditions_. Comparative phylogenetic analysis indicated a greater degree of shared evolutionary history between nosZ and nirS. However 30% of the organisms with nosZ did not possess either nir gene, with several of these also lacking nor, suggesting a potentially important role in N₂O reduction. Co-occurrence patterns were also non-randomly distributed amongst preferred habitat categories, with several habitats showing significant differences in the frequencies of nirS and nirK type denitrifiers. These results demonstrate that the denitrification pathway is highly modular, thus underpinning the importance of community structure for N₂O emissions.     

Genomics in a changing arctic: critical questions await the molecular ecologist

Molecular ecology is poised to tackle a host of interesting questions in the coming years. The Arctic provides a unique and rapidly changing environment with a suite of emerging research needs that can be addressed through genetics and genomics. Here we highlight recent research on boreal and tundra ecosystems and put forth a series of questions related to plant and microbial responses to climate change that can benefit from technologies and analytical approaches contained within the molecular ecologist's toolbox. These questions include understanding (i) the mechanisms of plant acquisition and uptake of N in cold soils, (ii) how these processes are mediated by root traits, (iii) the role played by the plant microbiome in cycling C and nutrients within high‐latitude ecosystems and (iv) plant adaptation to extreme Arctic climates. We highlight how contributions can be made in these areas through studies that target model and nonmodel organisms and emphasize that the sequencing of the Populus and Salix genomes provides a valuable resource for scientific discoveries related to the plant microbiome and plant adaptation in the Arctic. Moreover, there exists an exciting role to play in model development, including incorporating genetic and evolutionary knowledge into ecosystem and Earth System Models. In this regard, the molecular ecologist provides a valuable perspective on plant genetics as a driver for community biodiversity, and how ecological and evolutionary forces govern community dynamics in a rapidly changing climate.     

Effect of different carbon sources on the production of succinic acid using metabolically engineered Escherichia coli

Succinic acid (SA) is an important platform molecule in the synthesis of a number of commodity and specialty chemicals. In the present work, dual-phase batch fermentations with the E. coli strain AFP184 were performed using a medium suited for large-scale industrial production of SA. The ability of the strain to ferment different sugars was investigated. The sugars studied were sucrose, glucose, fructose, xylose, and equal mixtures of glucose and fructose and glucose and xylose at a total initial sugar concentration of 100 g L-1. AFP184 was able to utilize all sugars and sugar combinations except sucrose for biomass generation and succinate production. For sucrose as a substrate no succinic acid was produced and none of the sucrose was metabolized. The succinic acid yield from glucose (0.83 g succinic acid per gram glucose consumed anaerobically) was higher than the yield from fructose (0.66 g g-1). When using xylose as a carbon source, a yield of 0.50 g g-1 was obtained. In the mixed-sugar fermentations no catabolite repression was detected. Mixtures of glucose and xylose resulted in higher yields (0.60 g g-1) than use of xylose alone. Fermenting glucose mixed with fructose gave a lower yield (0.58 g g-1) than fructose used as the sole carbon source. The reason is an increased pyruvate production. The pyruvate concentration decreased later in the fermentation. Final succinic acid concentrations were in the range of 25-40 g L-1. Acetic and pyruvic acid were the only other products detected and accumulated to concentrations of 2.7-6.7 and 0-2.7 g L-1. Production of succinic acid decreased when organic acid concentrations reached approximately 30 g L-1. This study demonstrates that E. coli strain AFP184 is able to produce succinic acid in a low cost medium from a variety of sugars with only small amounts of byproducts formed.     

Community survey of ammonia-oxidizing bacteria in full-scale activated sludge processes with different solids retention time

AIMS: To study the effects of different solids retention time (SRT) on the nitrification activity and community composition of ammonia-oxidizing bacteria (AOB) in two full-scale activated sludge processes during a 5-month period. METHODS AND RESULTS: The AOB community composition was analysed using fluorescence in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE), and the identified populations were enumerated by quantitative FISH. Potential nitrification rates were determined in batch tests and the in situ rates were calculated from mass balances of nitrogen in the plants. Increased SRT reduced the nitrification activity, but neither the number per mixed liquor suspended solids nor community composition of AOB were affected. Two dominant AOB populations related to Nitrosomonas europaea and Nitrosomonas oligotropha were identified by FISH, whereas only the latter could be detected by DGGE. CONCLUSIONS: The effect of a longer SRT on the activity was probably because of physiological changes in the AOB community rather than a change in community composition. SIGNIFICANCE AND IMPACT OF THE STUDY: Physiological alterations of a stable AOB community are possible and may stabilize activated sludge processes. The commonly used FISH probes designed to target all beta-proteobacterial AOB does not detect certain Nitrosomonas oligotropha populations, leading to an underestimation of AOB if a wider set of probes is not used.     

Pancreatic Islet Survival and Engraftment Is Promoted by Culture on Functionalized Spider Silk Matrices

Transplantation of pancreatic islets is one approach for treatment of diabetes, however, hampered by the low availability of viable islets. Islet isolation leads to disruption of the environment surrounding the endocrine cells, which contributes to eventual cell death. The reestablishment of this environment is vital, why we herein investigated the possibility of using recombinant spider silk to support islets in vitro after isolation. The spider silk protein 4RepCT was formulated into three different formats; 2D-film, fiber mesh and 3D-foam, in order to provide a matrix that can give the islets physical support in vitro. Moreover, cell-binding motifs from laminin were incorporated into the silk protein in order to create matrices that mimic the natural cell environment. Pancreatic mouse islets were thoroughly analyzed for adherence, necrosis and function after in vitro maintenance on the silk matrices. To investigate their suitability for transplantation, we utilized an eye model which allows in vivo imaging of engraftment. Interestingly, islets that had been maintained on silk foam during in vitro culture showed improved revascularization. This coincided with the observation of preserved islet architecture with endothelial cells present after in vitro culture on silk foam. Selected matrices were further evaluated for long-term preservation of human islets. Matrices with the cell-binding motif RGD improved human islet maintenance (from 36% to 79%) with preserved islets architecture and function for over 3 months in vitro. The islets established cell-matrix contacts and formed vessel-like structures along the silk. Moreover, RGD matrices promoted formation of new, insulin-positive islet-like clusters that were connected to the original islets via endothelial cells. On silk matrices with islets from younger donors (<35 year), the amount of newly formed islet-like clusters found after 1 month in culture were almost double compared to the initial number of islets added.     

Identification of a plasminogen-binding motif in PAM, a bacterial surface protein

Surface-associated plasmin(ogen) may contribute to the invasive properties of various cells. Analysis of plasmin(ogen)-binding surface proteins is therefore of interest. The N-terminal variable regions of M-like (ML) proteins from five different group A streptococcal serotypes (33,41,52,53 and 56) exhibiting the plasminogen-binding phenotype were cloned and expressed in Escherichia coli . The recombinant proteins all bound plasminogen with high affinity. The binding involved the kringle domains of plasminogen and was blocked by a lysine analogue, 6-aminohexanoic acid, indicating that lysine residues in the M-like proteins participate in the interaction. Sequence analysis revealed that the proteins contain common 13–16-amino-acid tandem repeats, each with a single central lysine residue. Experiments with fusion proteins and a 30-amino-acid synthetic peptide demonstrated that these repeats harbour the major plasminogen-binding site in the ML53 protein, as well as a binding site for the tissue-type plasminogen activator. Replacement of the lysine in the first repeat with alanine reduced the plasminogen-binding capacity of the ML53 protein by 80%. The results precisely localize the binding domain in a plasminogen surface receptor, thereby providing a unique ligand for the analysis of interactions between kringles and proteins with internal kringle-binding determinants.